The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 80 kDa (predicted molecular weight: 80 kDa). For optimal results, primary antibody incubations should be performed at room temperature.
FunctionPhosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. May play a special role in the immune response. Protects cells against DNA damage-induced cell death.
Tissue specificityHighly expressed in spleen followed by thymus, peripheral blood leukocytes, pancreas, placenta. Weakly expressed in lung, kidney, prostate, ovary and colon.
Sequence similaritiesBelongs to the protein kinase superfamily. Ser/Thr protein kinase family. I-kappa-B kinase subfamily. Contains 1 protein kinase domain.
Post-translational modificationsAutophosphorylated. Sumoylation by TOPORS upon DNA damage is required for protection of cells against DNA damage-induced cell death. Desumoylated by SENP1.
Cellular localizationCytoplasm. Nucleus. Nucleus > PML body. Targeting to PML nuclear bodies upon DNA damage is TOPORS-dependent.
ICC/IF image of ab12142 stained Jeg3 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12142, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM
Western blot - IKKi/IKKe antibody [72B587] (ab12142)
Predicted band size : 80 kDa
IKK iota/IKK epsilon (80 kDa) detection by Western blot. The analysis of 10 µg of total cell extract from Daudi cells with the anti-IKK iota/IKK epsilon at 0.5 µg/ml (lane 1) and 3 µg/ml (lane 2) dilution.
IKKi/IKKen (80 kDa) detection by Western blot. The analysis of 10 µg of total cell extract from Daudi cells with the anti-IKK iota/IKK epsilon at 0.5 µg/ml (lane 1) and 3 µg/ml (lane 2) dilution.
Overlay histogram showing Jurkat cells stained with ab12142 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab12142, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-IKKi/IKKe antibody [72B587] (ab12142)
has not yet been referenced specifically in any publications.
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